The major goal of this proposal is to define the role of the protein SKI in human melanomas. SKI expression is highly elevated in melanomas compared to normal melanocytes. Their preliminary data demonstrates that SKI participates in the TGF-B signaling pathway and in the DNA damage response. He has found that SKI physically associates with the transcriptional co-activator proteins Smad2 and Smad3, two members of the TFG-B signaling pathway. In addition, forced expression of SKI results in inactivation of G1 checkpoints downstream from the protein p53. He proposes to determine the role of SKI in the TGF-B pathway and to determine the molecular mechanism(s) by which SKI interferes with the DNA-damage response in melanocytes and melanoma cells. Specifically, he will: (1) Define the molecular and biological activities of SKI in the TGF-G pathway by a) analyzing the activities of SKI/Smads complexes on TFG-B responsive promoters; b) determining whether SKI perturbs hetero-oligomer formation between Smad2 and Smad3 and the common-mediator Smad4; c) determining whether forced expression of SKI converts TGF-B-sensitive, normal human melanocytes to TGF-B-resistance and/or whether SKI represses TGF-B-mediated gene activation in vivo. (2) Analyze the molecular mechanism(s) whereby SKI suppresses p21 (Waf-1/SDI/Cip/1) expression. For that, he will determine whether; a) SKI interferes with the DNA-binding activity of the protein p53; b) SKI disrupts association of p53 with transcriptional activators; c) SKI interferes with Smad and p53 activity on the p21 promoter. 3) Explore the long-term consequences of SKI activity by determining whether forced expression of SKI results in chromosome instability in DNA-damaged cells. He will analyze whether these cells show a) abnormal centrosome amplifications; b) changes in Cyclin-Cdk-Cdk-I complexes that predispose cells to unrestricted growth. The studies proposed should underscore the role of SKI as a potent oncogenic protein in human melanomas and lay down foundations for future intervention therapies.